Long noncoding RNA TUG1 promotes proliferation, migration and cisplatin resistance in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a high metastasis rate and high recurrence rate, accounting for approximately 90% of oral cancers. Worldwide, there are more than 350,000 new cases each year. Approximately 50% of patients with OSCC are in an advanced stage at the time of treatment. Although there has been great progress in the diagnosis and treatment of tumors in recent years, the prognosis of OSCC has not been significantly improved. Studies have suggested that long noncoding RNAs (lncRNAs) are involved in the progression of cancer and can be used as cancer markers [1]. LncRNAs

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a high metastasis rate and high recurrence rate, accounting for approximately 90% of oral cancers. Worldwide, there are more than 350,000 new cases each year. Approximately 50% of patients with OSCC are in an advanced stage at the time of treatment. Although there has been great progress in the diagnosis and treatment of tumors in recent years, the prognosis of OSCC has not been significantly improved. Studies have suggested that long noncoding RNAs (lncRNAs) are involved in the progression of cancer and can be used as cancer markers [1].
LncRNAs are generally defined as RNA transcripts that are longer than 200 nucleotides and have no protein-coding potential. Approximately 4%-9% of the sequences in mammalian genomes produce transcripts as lncRNAs, while mRNA with protein coding function is only 1%. LncRNA is a kind of noncoding RNA transcript and it can regulate the level of miRNA and downstream gene expression, playing an important role in the regulation of gene expression and tumor formation, proliferation, invasion and metastasis [2]. With the research in depth on the molecular mechanism of OSCC occurrence and development, it shows that lncRNAs in OSCC are not only closely related to the clinicopathological characteristics of patients, but also affect the biological behavior of tumor cells by regulating miRNA or signaling pathways. It plays an important role in promoting or inhibiting cancer growth [3]. LncRNAs can not only be used as tumor diagnostic and prognostic factors, but also as tumor markers and targets for tumor therapy. Therefore, the study of differentially expressed lncRNAs is of great significance for the early diagnosis and treatment of OSCC.
In recent years, studies have shown that lncRNA TUG1 is highly expressed in lung cancer by Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) analyses [4]. In ovarian cancer, pancreatic cancer and other tumors, TUG1 is also involved in the regulation of cell proliferation, apoptosis and the EMT signaling pathway [5,6]. TUG1 possesses a 6.7-kb RNA sequence and regulates the cysteine derivative taurine, which is essential for neural development. TUG1 is highly expressed in many tumors, and the mechanism may be related to the formation of chromatin modification complexes and gene regulation. LncRNA TUG1 has been reported to activate the Wnt/β-catenin pathway. Overexpression of TUG1 induces doxorubicin (Dox) resistance in bladder cancer (BC) cell lines [7]. However, the knockout of TUG1 gene inhibited the resistance of BUC cells to Dox. It also demonstrated that TUG1 affects cell growth and drug-resistant SCLC. However, the expression and regulatory mechanism of TUG1 in OSCC are unclear. In this study, the expressions of TUG1 in OSCC tissues and cells were detected, the relationship between TUG1 expression and the prognosis of OSCC patients was analyzed, and the potential mechanism of TUG1 expression on the proliferation, migration, apoptosis and drug resistance of OSCC cells was explored.
To overexpress TUG1 protein, lentivirus vectors carrying TUG1 and the green fluorescent protein gene or the TUG1 gene alone were successfully constructed. TUG1 and green fluorescent protein were successfully expressed in human tongue squamous cell carcinoma cell line SCC9 (Hunan Fenghui Biotechnology Co., Ltd, Changsha, China), compared with cells infected with the lentivirus empty vectors which were used as the negative control (NC). Meanwhile, knockdown of TUG1 was achieved by transfection of SCC9 cells with si-TUG1 (5′-GGUUGGUUGUGGGAUUUCUTT-3′; GenePharma, Shanghai, China), using si-NC (Cat No. A01001; GenePharma) as the negative control.
The optimal MOI was 40 at 72 h after infection with lentivirus carrying TUG1 and green fluorescent proteins (data not shown). The level of TUG1 in the TUG1-OE group was significantly higher than that in the control (NC) group, and the level of TUG1 in the si-TUG1 group was significantly lower than that in the si-NC group ( Figure 1A). The transwell assay results showed that the number of migrated cells in the TUG1-OE group was higher than that in the control groups (P<0.001), and the number of migrated cells in the si-TUG1 group was lower than that in the si-NC group (P<0.001; Figure 1B). These results suggested that lncRNA TUG1 significantly promoted SCC9 cell migration in vitro. To further verify the role of lncRNA TUG1 in SCC9 cell apoptosis, the cell apoptosis rate was detected by Annexin V/PI staining and flow cytometry. The results showed that cell apoptosis was increased in the si-TUG1 group compared with that in the si-NC group, and cell apoptosis was decreased in the TUG1-OE group compared with that in the control group ( Figure 1C).
CKK-8 assay was performed to explore the viability of SCC9 cells. The cell viability of SCC9 cells was inhibited after12 h, 24 h and 48 h of treatment with increasing concentrations of cisplatin. The death rate was <50% when the cells were treated with 4 μM cisplatin for 72 h (Table 1). After treatment with cisplatin (4 μM) for 12 h, 24 h and 48 h, cell viability was decreased, and apoptosis was increased with time. The cell viability of the TUG1-OE group was significantly higher than that of the NC and si-TUG1 groups (P<0.001). The cell viability of the si-NC group was significantly higher than that of the si-TUG1 group (P<0.001) and the cell viability gradually decreased within 48 h (Figure 2A-C). The OD 450nm value of the TUG1-OE   Figure 2D). The apoptosis rate in the TUG1-OE group was lower than that in the NC group after treatment with cisplatin for 12 h, 24 h and 48 h. In contrast, the apoptosis rate in the si-TUG1 group was higher than that in the si-NC group after treatment with cisplatin for 12 h, 24 h and 48 h ( Figure 2E). Our transwell assay results also showed that cell migration of the TUG1-OE group was 1.5~2-fold higher than that of the control group. Cisplatin could obviously inhibit the proliferation of SCC9 cells and promote cell apoptosis. Knockdown of TUG1 in cisplatin-treated SCC9 cells significantly inhibited cell growth, suggesting that TUG1 could promote drug resistance, migration and proliferation of SCC9 cells. TUG1 lncRNA acts as a sponge to adsorb downstream targets involved in drug resistance and downstream genes by changing the activity of transcription factors related to drug resistance genes. Although the results proved that TUG1 regulated OSCC drug resistance, the mechanism is still unclear. The molecular mechanism by which lncRNA TUG1 promotes the drug resistance of OSCC cells still needs to be explored in further studies. The DLX1, P53, and β-catenin are the key factors in cell apoptosis pathways, and overexpressions of DLX1, NF-κB, and β-catenin inhibit cell apoptosis and promote cell proliferation [8]. TNF-α and P53 promote cell apoptosis and inhibit cell proliferation [9]. Our results showed that DLX1, NF-κB, and β-catenin in the TUG1-OE group were significantly higher than those in the control group, and TNF-α and P53 expressions were significantly lower than those in the control group (P<0.01). DLX1, NF-κB, and β-catenin in the si-TUG1 group were significantly lower than those in the si-NC group, while TNF-α and P53 were significantly higher than those in the si-NC group (P<0.01) (Supplementary Figure S1A,B). These results suggest that TUG1-OE promotes SCC9 cell proliferation and inhibits cell apoptosis. In contrast, si-TUG1 promotes SCC9 cell apoptosis and inhibits cell proliferation.
In summary, in the current study we found for the first time that lncRNA TUG1 is significantly overexpressed in SCC9 cells. High expression of lncRNA TUG1 could promote cell proliferation and cell migration, inhibit cell apoptosis, and promote malignant biological behaviors such as drug resistance, indicating that TUG1 may play an important role in the process of cell proliferation and apoptosis during the occurrence and development of OSCC. This study may provide a new idea for targeted therapy of OSCC.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

Funding
This work was supported by the grants from the Zhejiang